Seed development occurs in three overlapping phases. Initially a developmental pattern for embryo body plan and supporting tissue is established and elaborated, followed by synthesis of nutrient reserves for future use in nursing a germinating embryo to its autotrophic state, and finally preparation for the existence of a seed as a dormant entity awaiting favourable conditions for its next life cycle (Thomas 1993; Bewley and Black 1994; Goldberg et al., 1994; Wobus and Weber, 1999). Even after an embryo has attained its ability to potentially grow into a plant, the so-called maturation phase continues and then culminates in metabolic quiescence. This preparative phase includes progressive desiccation to retain in fully mature seeds a water content of only 5-15% that is sufficient to maintain cellular integrity but not to sustain normal biochemical activity. This phenomenon contrasts so-called orthodox seeds (storable as dry seeds) from seeds that are recalcitrant to storage (Farnsworth, 2001). Recalcitrant seeds do not dehydrate during their development but instead proceed to germinate, and in fact do not survive if dehydration is imposed. Vivipary, a variation in which embryos germinate prior to dispersal from their parent, also lacks a dehydration phase. Some consider embryogenesis-to-germination as a primitive feature into which desiccation has been interposed subsequently. However, there is no clear-cut pattern and the ability to desiccate is possibly an acquisition that occurred at multiple times in various taxa (Pammenter and Berjak, 2000). The uncertainty of the evolutionary chronology of the phenomenon aside, the ability of a seed to desiccate—and the cellular events pertinent to it—can impact pre-harvest physiology and post-harvest characteristics such as seed storage and vitality.
During seed maturation phase, proteins unrelated to major storage reserves also accumulate and continue to do so long after the storage genes cease their transcription. Initially found in wheat, and referred to as early methionine-labelled protein gene (Em) (Grzelczak et al., 1982; Cuming, 1984), related late embryogenesis abundant (LEA) proteins were recognized formally as a new group of proteins in cotton seeds (Baker et al., 1988). These are also structurally related to some of the proteins identified independently as being associated with responses to abiotic stress factors such as cold, dehydration and salinity, and abscisic acid treatment in vegetative parts and seed tissue ((Dure et al., 1989; Mundy et al., 1990; Bray, 1994; Close et al., 1993; Thomas, 1993; Dure, 1997). Following the discovery of LEA proteins in other species, Dure et al (1989) employed predicted structural features of the LEA proteins to classify them into three typical groups, and later into at least five groups (Bray, 1994; Moons et al., 1997; Cuming, 1999). All the groups have some typical common features such as high overall hydrophilicity and general paucity of Cys and Trp residues (Dure, 1997). Characteristically, Most of these proteins do not coagulate on boiling and remain soluble hence the use of the term “boiling-stable” in the literature.
Various groups of LEA proteins are also synonymously referred to with a “D-numeral”, for example Group 1 as D19 family, which signifies the relationship to the embryogenesis-associated cDNA clones of cotton seeds (Dure, 1997). Group 1 LEA proteins have a 20-aa segment that is repeated 2 to 4 times in some species. The Em protein of wheat is a classical member of this group (Grzelczak et al., 1982; Quatrano et al. 1997). The group members are rich in Gly, are highly hydrated, and have random coil conformation (McCubbin and Kay 1985). Group 2 LEA (D11 family), the largest class, includes proteins that have been referred to under various names such as dehydrins, COR and RAB, and shows a wide variation in polypeptide size (9 to 200 kDa) (Bray et al., 1994; Close, 1997; Thomashow, 1999). The biological contexts in which these proteins were identified are quite diverse: dehydrating leaves, ABA-treated vegetative tissue, desiccating embryo, cold- or salinity-stressed tissues. The Group 2 proteins have one to 11 copies of a Lys-rich 15-mer unit (K-domain), and additionally either a Y-domain near the N-terminus or a poly-Ser domain or both. These proteins are also largely amorphous. Group 3 LEA (D7) proteins are dealt with after Group 4 (D113). The latter contains structurally bipartite proteins that have a Lys- and Glu-rich helical region followed by a Gly-rich random coil of highly variable length among different members (Ingram and Barterls, 1996).
The common feature of Group 3 Lea proteins is the presence of a 11-aa repeat in the polypeptide (Dure et al., 1989). Members of this group include cotton D7 (Baker et al., 1988), barley HVA1 (Straub et al., 1994), carrot Dc3 (Seffens et al., 1990) and Dc8 (Dure, 1993a), and rapeseed LEA76 (Harada et al. 1989). The 11-mer is a motif in which all but Position 10 contain an aa of specific chemical characteristic. Positions 1, 2, 4, 5 and 9 have mostly Ala, Thr, and to a lesser extent Val, aa that have side chains terminating in methyl group, and Positions 3, 7 and 11 have polar aa Glu, Gln, or Asp, and Positions 6 and 8 have positively charge aa Lys or Arg (Dure, 1993). The 11-mer repeats are predicted to result in an amphipathic helices in which one face contains the methylated aa in hydrophobic stripe and the other contains polar and charged aa; potential ionic bridges, for example, between Lys of Position 8 and Glu of Position 11, are considered to stabilize the helix. The remarkable positional conservation of hydrophobic, polar and charged aa does indicates a functional constraint despite the taxonomic divergence of the species where these proteins occur.
All LEA proteins have a common physiological context. Their production is associated with intracellular desiccation caused by or associated with various developmental (seed maturation), hormonal (ABA) and environmental conditions (drought, salinity, cold). Although ABA is intimately associated with cellular dehydration, ABA-induced LEA gene expression in vegetative tissues varies from undetectable (Prieto-Dapena et al., 1999) to high (Straub et al., 1994).
Jasmonic acid (JA) is involved in various processes including wounding response, senescence, pathogen attack, fruit removal and under dehydration (Creelman and Mullet, 1995). JA also interacts with ABA in modulating ABA-inducible gene transcription (Hays et al., 1999). Recently, Swiatek et al (2002) reported that JA and ABA regulated cell division progression differentially.
Cellular dehydration is countered with accumulation of compatible solutes such as glycine betaine (Jain and Selvaraj, 1997; Nuccio et al., 1999) to a certain extent, but extensive loss of bulk water and especially bound water can lead to irreversible damages of oraganelles, membranes, proteins and enzymes. The concomitant increase of ion concentration would worsen the intracellular conditions. The coincidental high-level accumulation of LEAs under developmental and environmental dehydration, intracellular location, and the generally amorphous structure and the very high water-carrying capacity of LEAs have favored a non-enzymatic role for LEAs in safeguarding the cellular entities. These include hydration, water substitution in hydrogen bonding to membrane polar heads, and acquisition of a glassy state, and ion sequestration (Bartels et al., 1988; Lane 1991; Close et al., 1993; Ried and Walker-Simmons, 1993; Dure, 1997; Thomashow, 1999). Group 3 LEAs, because of their 11-aa modules of predicted helices, are considered to prevent salt precipitation and deleterious crystal formation by trapping ions (Dure, 1997). Interestingly, LEA3 proteins from pollen can stabilize glasses in vitro due to the possible formation of tight hydrogen bonding network with sugars (Wolkers et al., 2001). As evident from the foregoing, groups of unstructured proteins with lax primary and higher order organization cannot be collectively understood by paradigms since there is no paradigm per se for such proteins, but the examples do provide a starting point. The accumulation of Group 3 LEA proteins correlates with dehydration tolerance in wheat seedlings (Ried and Walker-Simmons, 1993) and cold acclimation efficiency in rice (Takahashi et al., 1994) and in Chlorella (Joh et al., 1995). Ectopic expression of the barley LEA3 (HVA1) in rice (Xu et al., 1996), tomato LE25 protein in yeast (Imai et al., 1996) result in enhanced drought and salt tolerance in transgenic rice, salt and freezing tolerance in yeast, respectively. Brassica napus (rapeseed; canola) is an economically important seed crop. The potential importance of LEA3 in seed development is not known. With the exception of an early report by Harada et al (1989) on LEA76 cDNA from a library of germinating seeds (14 hr after imbibition), there is very little information. The organization of LEA3 gene family or the promoter characteristics of its members have not been determined.